Second Additional Phytase Variants and Methods

ABSTRACT

The present invention relates to variant phytase enzymes and their use thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 7, 2021 is named 114095-5013-US_ST25.txt and is 22 kilobytes in size.

BACKGROUND OF THE INVENTION

Phytate is the major but indigestible form of phosphorus found in plant-based feeds. It is considered as an anti-nutritional factor (ANF) that needs to be reduced or removed from cereal-based foods and feeds. Under acidic conditions, phytate interacts with positively charged dietary proteins leading to the formation of phytate-protein aggregates and precipitates, which results in a decreased accessibility for proteases, and consequently in inefficient protein digestion. Phytate also acts as a strong chelating agent that binds different vital metal ions in foods and feeds in the small intestine of monogastric organisms, leading to nutritional deficiencies of many important minerals like calcium, zinc, etc. in animals.

Phytase is a phosphatase that catalyzes the hydrolysis of O—P bonds in phytate and releases inorganic usable phosphorous. Phytase plays versatile roles in agricultural and feeding fields. Ruminant animals such as cattle and sheep can utilize the phytate in grains as a source of phosphorus since they have bacteria in the gut that produces phytases. Non-ruminants like pigs, poultry, fish, dogs, birds, etc. require extrinsic phytase to liberate inorganic phosphorous. Hence, addition of inorganic phosphorous, a non-renewable and expensive mineral, to feeds for monogastric animals is a common practice, which incurs heavy costs to the feed industry. Consequently, phytase produced from various sources have emerged as one of the most effective and lucrative supplement to these species' diets to enhance the nutritional value of animal feeds and decrease animals' phosphorus excretion that leads to environmental pollution.

Phytase in feeds can be inactivated by temperature during feed processing (pelleting), by the low pH or pepsin in the upper part of the gastrointestinal tract of an animal. Selle and Ravindran laid out the characteristics for an ideal feed enzyme, namely; 1) a high specific activity per unit of protein, 2) good thermostability during feed processing, 3) high activity in the typical pH range of the animal gut, 4) resistance to gastric proteases, and 5) good stability under ambient temperatures. (SELLE, P. H. and RAVINDRAN, V. (2007) Microbial phytase in poultry nutrition. Animal Feed Science and Technology 135: 1-41.)

The heat treatment of feeds can involve heat alone or a combination of both heat and pressure. The most common form of thermal treatment in the manufacture of poultry feeds is pelleting. The pelleting process first involves the mash feed passing through a conditioner. In the conditioner the cold feed is exposed to dry steam which is added under pressure. This process helps to improve pellet durability and also increases mill throughputs and reduces energy consumption. Under these conditions, plant cells are crushed, which is favorable for the digestion process in animals. Nissinen found that moderate conditioning less than 85° C. was optimal for broiler performance and high conditioning temperature at 95° C. resulted in poorer body weight gain and feed conversion ratio (NISSINEN, V. (1994) The effects and interactions of enzymes and hydrothermal pre-treatments and their contribution to feeding value. International Milling Flour and Feed, May: 21-22). Pelleting process at 65-85° C. usually result in improving the availability of nutrients due to the rupture of the cell wall matrix (PICKFORD, J. R. (1992) Effects of processing on the stability of heat labile nutrients in animal feeds, in: GARNSWORTHY, P. C., HARESIGN, W. & COLE, D. J. A. (Eds) Recent Advances in Animal Nutrition, pp. 177-192 (Butterworth-Heinemann, Oxford, U.K.) and deactivation of enzyme inhibitors present in cereals (SAUNDERS, R. M. (1975) α-Amylase inhibitors in wheat and other cereals. Cereal Foods World 20: 282-285). The effect of the damage on the phytase activity due to high pressure appears to be small; it is mainly the high temperature which results from the high energy input that inactivates the enzyme. Thus, developing a thermostable Phytase provides an attractive solution for cost-effective processes in the feed industry.

New applications of phytase in human foods are similarly important as those in animal feeds because indigestible phytate chelates essential minerals and contributes to deficiencies of these nutrients in approximately two to three billion people. The applications of phytase in human health and medicine represent other new exciting areas. In addition, phytase has great potentials for industrial applications including food processing and biofuel production. Thermostable phytase, along with xylanase, have been suggested as effective additives in the pulp and paper industry.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides variant phytases and methods of producing and using them. The amino acid sequence numbers of the present invention are listed in Table 1.

TABLE 1 Amino acid sequence numbers and nucleic acid sequence numbers. G7P/CL00100223 mature protein SEQ ID NO: 1 G7V1/G8P mature protein SEQ ID NO: 3 G9V1 mature protein SEQ ID NO: 5 G1P mature protein SEQ ID NO: 7 N.A. encoding G7P/CL00100223 mature protein SEQ ID NO: 2 N.A. encoding G7V1/G8P mature protein SEQ ID NO: 4 N.A. encoding G9V1 mature protein SEQ ID NO: 6 N.A. encoding G1P mature protein SEQ ID NO: 8

In one aspect, the invention provides compositions comprising a variant phytase enzyme comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1; and wherein said variant phytase enzyme has phytase activity.

In another aspect, the invention provides compositions comprising a variant phytase enzyme comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 2-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.; and wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1.

In a further aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said variant phytase enzyme has at least 90% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1.

In another aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said variant phytase enzyme has at least 10-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.

In an additional aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said variant phytase enzyme comprises one or more amino acid substitutions as compared to SEQ ID NO:1 and has at least 90% sequence identity to SEQ ID NO:1.

In another aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said variant phytase enzyme has at least 2% increase in residual activity as compared to SEQ ID NO:1 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.

In a further aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said one or more amino acid substitution(s) of SEQ ID NO:7 occur at one of said positions, two of said positions, three of said positions, four of said positions, five of said positions, six of said positions, seven of said positions, eight of said positions, nine of said positions, ten of said positions, eleven of said positions, twelve of said positions, thirteen of said positions, fourteen of said positions, fifteen of said positions, sixteen of said positions, seventeen of said positions, eighteen of said positions, nineteen of said positions, twenty of said positions, twenty-one of said positions, twenty-two of said positions, twenty-three of said positions, twenty-four of said positions, twenty-five of said positions, twenty-six of said positions, twenty-seven of said positions, twenty-eight of said positions, twenty-nine of said positions, thirty of said positions, thirty-one of said positions, thirty-two of said positions, thirty-three of said positions, thirty-four of said positions, thirty-five of said positions, thirty-six of said positions, thirty-seven of said positions, thirty-eight of said positions, thirty-nine of said positions, forty of said positions, forty-one of said positions, forty-two of said positions, or forty-three of said positions of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7 or SEQ ID NO:1.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N, K7N, A25F, A25N, A25Q, D35N, I55V, H60Q, V67A, D69N, A73P, K74D, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, S120R, P121A, D136S, N137P, G157Q, R159Y, P173N, Q192N, S212P, T238G, Y255D, M276K, A277T, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, A288S, E315G, Q346S, F354Y, T364W, T378A, A380P, G381A, and A403G of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzyme as disclosed herein, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288S/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284V/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281T/H282Q/P283G/P284A/Q285T/K286E/Q287N/A288K/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25Q/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25F/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S119G/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/Q192N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, and 212 of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, and S212P of SEQ ID NO:7.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or two further amino acid substitutions occurring at positions corresponding to positions 74 and/or 192 of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said one or two further amino acid substitutions are K74D and/or Q192N.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 120, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.

In an additional aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5.

In an additional aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises the sequence of SEQ ID NO:3.

In a further aspect, the invention provides compositions comprising the variant phytase enzymes as disclosed herein, wherein said variant phytase enzyme comprises the sequence of SEQ ID NO:5.

In an additional aspect, the invention provides the composition comprising the variant phytase enzymes as disclosed herein, wherein the composition further comprises animal feed.

The invention further relates to polynucleotide comprising nucleotide sequences which encode the phytase variants produced by the method, nucleic acid constructs comprising the polynucleotides operably linked to one or more control sequences that direct the production of the polypeptide in an expression host, recombinant expression vectors comprising such nucleic acid constructs, and recombinant host cells comprising a nucleic acid construct and/or an expression vector.

In one aspect, the invention provides a nucleic acid encoding the variant phytase enzyme as disclosed herein.

In a further aspect, the invention provides the nucleic acid as disclosed herein, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:4.

In an additional aspect, the invention provides the nucleic acid as disclosed herein, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:6.

In a further aspect, the invention provides an expression vector comprising the nucleic acid as disclosed herein.

In an additional aspect, the invention provides a host cell comprising the nucleic acid as disclosed herein or the expression vector as disclosed herein.

In a further aspect, the invention provides a method of making a variant phytase enzyme comprising (a) culturing the host cell as disclosed herein under conditions in which said variant phytase enzyme is produced, and (b) recovering said enzyme.

In an additional aspect, the invention provides variant phytase enzymes having an amino acid substitution set selected from the group consisting of those depicted in FIGS. 1-3 .

In some aspects, the invention relates to phytase variants having improved thermal properties, such as thermostability, heat-stability, Bovine Serum Albumin (BSA) stability, pH stability, steam stability, temperature profile, and/or pelleting stability, with variant enzymes having high tolerance to high temperature and/or high tolerance to low pH of particular use in many embodiments.

In additional aspects, the invention relates to phytase variants having improved pelleting stability and/or improved acid-stability.

The method of the invention thus relates to phytase variants having improved protease stability, in particular pepsin stability, found in non-ruminant stomachs.

The method of the invention thus relates to phytase variants having improved performance in animal feed (such as an improved release and/or degradation of phytate).

In a further aspect, the invention relates to methods for improving the nutritional value of an animal feed, by adding a phytase variant of the invention to the feed, processes for reducing phytate levels in animal manure by feeding an animal with an effective amount of the feed, methods for the treatment of vegetable proteins, comprising the step of adding a phytase variant to at least one vegetable protein, and the use of a phytase variant of a composition of the invention.

The invention also provides a method for producing a fermentation product such as, e.g., ethanol, beer, wine, comprising fermenting a carbohydrate material in the presence of a phytase variant, a method for producing ethanol comprising fermenting a carbohydrate material in the presence of a phytase variant and producing ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show thermostability results of G1P wild type phytase and G7-G9 phytase variants produced by Saccharomyces cerevisiae. FIG. 1A shows thermostability results of G1P wild type phytase and phytase variants: G7P, G7V1 (G8P), G7V2, G7V3, G7V4, G7V5 and G7V6. “—” means that signal is too low to be determined accurately. FIG. 1B shows thermostability results of phytase variants: G7V7, G7V8, G7V9, G7V10, G7V11, G8V1, and G8V2. FIG. 1C shows thermostability results of phytase variants: G8V3, G8V4, G8V5, G8V6, G8V7 and G9V1.

FIG. 2 provides a summary of phytase variants with reference to G1P.

FIGS. 3A-3D provide sequence listings. FIG. 3A provides the amino acid sequence of G7P/CL00100223 mature protein (SEQ ID NO:1) and DNA sequence (SEQ ID NO:2) encoding G7P/CL00100223 mature protein. FIG. 3B provides the amino acid sequence of G7V1/G8P mature protein (SEQ ID NO:3) and DNA sequence (SEQ ID NO:4) encoding G7V1/G8P mature protein. FIG. 3C provides the amino acid sequence of G9V1 mature protein (SEQ ID NO:5) and DNA sequence (SEQ ID NO:6) encoding G9V1 mature protein. FIG. 3D provides the amino acid sequence of G1P mature protein (SEQ ID NO:7) and DNA sequence (SEQ ID NO:8) encoding G1P mature protein.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Phytases decompose phytate (inositol hexakisphosphate (IP6) or phytic acid when in the salt form), which is the primary storage of phosphate in plants. Monogastric animals such as swine, poultry and fish (as well as humans) cannot digest phytate, leading to phosphorus excretion in the manure, which poses an environmental concern in agricultural areas. In addition, the phytate can lead to aggregation of proteins, reducing protein availability, as well as chelate minerals and trace elements, further reducing the available nutrients for the animals.

The addition of phytase to animal feed was introduced several decades ago and can reduce phosphorus excretion by up to 50% while also allowing the animal better access to the available nutrients. However, under the conditions which are used in the processing of many foods, including both animal feeds made from plant sources as well as foods for humans (cereals, etc.) such as higher temperatures and different pHs, many wild type phytases are not very stable, leading to inefficient conversion of the phytate and/or the cost prohibitive addition of more enzyme. Similarly, other uses for phytase such as in the production of biofuels also can include higher temperatures and/or different pHs. Accordingly, it is an object of the present invention to provide variant phytases with improved properties, including thermostability and other biochemical properties as outlined herein, that lead to improved outcomes such as less environmental stress due to lowered phosphorus excretion, better feed conversion to animal weight and better access to nutrients.

II. Definitions

By “modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. By “amino acid modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution I55V refers to a variant polypeptide, in this case a phytase, in which the isoleucine at position 55 is replaced with valine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example, exchanging CGG (encoding arginine) with CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, −233E or 233E designates an insertion of glutamic acid after position 233 and before position 234. Additionally, −233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233 #, E233( ) or E233del designates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233 #designates a deletion of the sequence GluAspAla that begins at position 233.

By “parent polypeptide” as used herein is meant a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it. In the present case, some embodiments utilize wild type G1P (SEQ ID NO:7), G7P (SEQ ID NO:1), G8P (SEQ ID NO:3), or G9V1 (SEQ ID NO:5) as parent polypeptides.

By “variant protein” or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification. Protein variant may refer to the protein itself, a composition comprising the protein, or the amino sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, e.g. from about one to about seventy amino acid modifications, and preferably from about one to about forty-five amino acid modifications compared to the parent. As described below, in some embodiments, the parent polypeptide is a wild type sequence, for example, having the amino acid sequence of SEQ ID NO:7. As further discussed below, the protein variant sequence herein will preferably possess at least about 80% identity to a parent protein sequence, and most preferably at least about 90% identity to the parent polypeptide. In some embodiments, the protein variant sequence possesses at least about 95%, 98%, or 99% identity to a parent polypeptide. Variant protein can refer to the variant protein itself, compositions comprising the protein variant, or the DNA sequence that encodes it. Thus, by “variant phytase” herein is meant a novel phytase that has one or more amino acid modifications in the amino acid sequence as compared to a parent phytase enzyme. As discussed herein, in some cases the parent phytase is a higher generation of variant; that is, as shown in FIG. 1A, the G7P phytase has 27 amino acid substitutions P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G as compared to the wild type G1P parent. However, the G8P has 9 amino acid substitutions T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R as compared to the G7P parent, but a total of 36 amino acid substitutions as compared to the G1P (SEQ ID NO:17). Unless otherwise noted or as will be obvious from the context, the variant phytases of the invention generally are compared to the G1P or G7P sequence. Additionally, unless otherwise noted, the variant phytases of the invention are enzymatically active, that is, there is detectable phytase activity using the phytase assay described in the Examples.

As used herein, “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The peptidyl group generally comprise naturally occurring amino acids and peptide bonds. In addition, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and its associated amino acid identity. For example, Histidine 82 (also referred to as His82 or H82) is a residue at position 82 in the G1P wild type enzyme.

By “non-naturally occurring modification” as used herein is meant an amino acid modification that is not found in the wild type (e.g. G1P) enzyme.

By “amino acid” and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.

By “position” as used herein is meant a location in the sequence of a protein. In general, the position number (which is more fully discussed below) is relative to the first amino acid of the mature phytase sequence, e.g. excluding the signal peptide.

By “phytase” herein is meant a protein with phytase activity. By “phytase activity” herein is meant that the enzyme catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to (P6689oldab*1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate. Enzymes having detectable activity in the assay outlined below and in the Examples are considered phytases herein.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity” or “identity”. The degree of identity between an amino acid sequence of the present invention (“invention sequence”) and the parent amino acid sequence referred to in the claims (e.g. G1P, SEQ ID NO:7 or G7P, SEQ ID NO:1) is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the “invention sequence,” or the length of the parent amino acid sequence, whichever is the shortest. The result is expressed in percent identity as calculated below.

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO:7 is used to determine the corresponding amino acid residue in another phytase of the present invention. The amino acid sequence of another phytase is aligned with the mature polypeptide disclosed in SEQ ID NO:7, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:7 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Identification of the corresponding amino acid residue in another phytase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), and EMBL-EBI employing Clustal Omega (Sievers and Higgins, 2014, Methods Mol Biol. 2014; 1079:105-16), using their respective default parameters.

When the other enzyme has diverged from the parent polypeptide such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The standardly accepted IUPAC single letter or three letter amino acid abbreviation is employed.

For an amino acid substitution, the following nomenclature is used herein: Original amino acid, position, substituted amino acid. Accordingly, the substitution of proline at position 4 with asparagine is designated as “Pro4Asn” or “P4N”. Multiple mutations are separated by forward slash marks (“/”), e.g., “P281T/H282N/P283G”, representing substitutions at positions 281, 282 and 283, respectively.

Abbreviation 1 letter abbreviation Amino acid name Ala A Alanine Arg R Arginine Asn N Asparagine Asp D Aspartic acid Cys C Cysteine Gln Q Glutamine Glu E Glutamic acid Gly G Glycine His H Histidine Ile I Isoleucine Leu L Leucine Lys K Lysine Met M Methionine Phe F Phenylalanine Pro P Proline Ser S Serine Thr T Threonine Trp W Tryptophan Tyr Y Tyrosine Val V Valine

By “isolated” in the context of a phytase herein is meant that the polypeptide is devoid of other proteins. In a particular embodiment the phytase of the invention is isolated. The term “isolated” as used herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95 to 98% pure, as determined by SDS-PAGE. In particular, it is preferred that the polypeptides are in “essentially pure form”, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods.

By “recombinant enzyme” herein is meant that the enzyme is produced by recombinant techniques and that nucleic acid encoding the variant enzyme of the invention is operably linked to at least one exogeneous (e.g. not native to the parent phytase) sequence, including, for examples, promoters, terminators, signal sequences, etc., as are more fully outlined below.

The term “nucleic acid construct” refers to a nucleic acid molecule, either single-stranded or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences.

The term “operably linked” refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

III. Phytases of the Invention

Accordingly, the present invention provides variant phytases with improved activity that can be used in a variety of applications, including animal and human nutritional and feed products and the production of biofuels such as bioethanol.

In general, the variant phytases of the invention have modified, improved biochemical properties as compared to the wild type G1P phytase (SEQ ID NO:7), and/or the Generation 7 variant phytases, (i.e. G7P, SEQ ID NO:1) as shown in FIGS. 1 and 2 . The biochemical properties of the variant phytases that can be improved herein include, but are not limited to, thermostability, residue activity, pH activity, pH stability, specific activity, formulation stability (including liquid, solid and pellets), performance in animals and/or animal feed and protease stability.

The variant phytases of the invention have one or more improved properties as compared to G1P and/or G7P. By “improved” herein is meant a desirable change of at least one biochemical property. “Improved function” can be measured as a percentage increase or decrease of a particular activity, or as a “fold” change, with increases of desirable properties (e.g. thermostability or residual activity) or decreases of undesirable properties (e.g. protease sensitivity). That is, a variant phytase may have a 10% increase in residual activity after thermochallenge or a 10% decrease in protease sensitivity, as compared to a parental phytase. Alternatively, a variant phytase may have a 2-fold increase in pH stability or a 3-fold decrease in protease sensitivity. By “residual activity” herein meant the enzymatic activity of an enzyme after thermochallenge/heat treatment as compared with the activity of the same enzyme placed under room temperature for the same period of time without thermochallenge/heat treatment. The activity of the untreated sample and the heat-treated samples were determined by the phytase enzymatic assay mentioned below, and the % residual activity was calculated as Activity_(Heat-treated sample)/Activity_(Untreated sample)×100%. In general, percentage changes are used to describe changes in biochemical activity of less than 100%, and fold-changes are used to describe changes in biochemical activity of greater than 100% (as compared to the parental enzyme, in many cases, G1P and/or G7P). In the present invention, percentage changes (usually increases) of biochemical activity of at least about 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and 99% can be accomplished. In the present invention, a “fold increase” (or decrease) is measured as compared to the starting or parent enzyme. For example, as shown in the FIG. 1A, G7V6 has a 1.08 fold increase in residual activity after thermochallenge at about 85° C. as compared to G7P: this is calculated by [(residue activity of variant)/(residual activity of parent)]. In many embodiments, the improvement is at least 1.01 fold, 1.05 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold, 15 fold or higher.

In general, improvements in thermostability of variant phytases are measured as compared to a parental phytase enzyme using a phytase enzymatic assay disclosed below, under the same challenge conditions.

A. Phytase Assay

The phytase enzymatic assay to determine thermostability is run as shown in Example 3, and as follows: the supernatants comprising EcPhytase Produced by Saccharomyces cerevisiae in HTP (e.g. from Example 2) are 100 fold diluted using 0.25M sodium acetate pH 5.5. 50 μl of the diluted supernatants are transferred to PCR plates and heated at 75° C., 80° C., 85° C. and 90° C. for 5 minutes in thermocyclers and subsequently cooled on ice for 2 minutes. In 96 well shallow microtiter plates, 50 μl of the untreated supernatants and heat-treated supernatants are separately added to 100 μl of sodium phytate substrate (C₆H₆Na₁₂O₂₄P₆, FW: 923.81) prepared in 0.25M sodium acetate, pH 5.5. The reaction is incubated at 37° C., 150 rpm for 30 minutes. The reaction is quenched with 100 μl of stop solution (1:2 nitric acid, 100 g/L ammonium molybdate, 2.35 g/L Ammonium vanadate). After the plates are shaked for 30 seconds, they are subjected to plate reader for reading at 415 nm. The activity of the untreated sample and the heat-treated samples are determined by the activity analysis method mentioned above, and the % residual activity is calculated as Activity_(Heat-treated sample)/Activity_(Untreated sample)×100%. The residual activity of a variant enzyme was compared to the parent enzyme under the same conditions to determine activity improvement. In some cases, it is useful to use “Phytase Units”, or PU, defined as the amount of phytase required to liberate 1 μmol of inorganic phosphate per minute. The enzyme may be a purified sample, a fermentation sample, or a raw sample.

The variant phytases of the invention can have an improvement in one or more of a number of biochemical properties, including, but not limited to, thermostability, pH activity, pH stability, specific activity, formulation stability (including liquid, solid and pellets), BSA stability, performance in animals and/or animal feed and/or protease stability.

B. Thermostability

In many embodiments, the variant phytases of the invention have increased thermostability, particularly under the conditions used to produce animal feeds, for example, which frequently use high temperatures during the pelleting process for periods of time that traditionally inactivate wild type phytases. “Thermostability” in this context means that the variant enzymes are more stable than the parental phytase (e.g. G1P or G7P) under the same thermal challenge conditions, that is, the residual activity of the variant is higher than that of the parental phytase under identical conditions (generally using the phytase enzymatic assay as outlined herein and as shown in Examples).

In one embodiment, the variant phytases are more stable than the parent phytase when exposed to temperatures of 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C. and/or 90° C. for a period of time, generally ranging from about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes or longer, depending on the ultimate conditions for the use of the variant phytase, with some embodiments utilizing thermal challenge times of 5 minutes to 10 minutes, 5 minutes to 15 minutes, 5 minutes to 60 minutes, 10 minutes to 60 minutes all finding use in the present invention. In some embodiments, a challenge of 75° C., 80° C., 85° C. or 90° C. is used.

Accordingly, in some embodiments the variant phytases have increased thermostability with or without the presence of BSA as compared to a parent phytase, e.g. G1P and/or G7P, for at least 5-10 minutes at 55° C., at least 5-10 minutes at 65° C., at least 5-10 minutes at 70° C., at least 5-10 minutes at 75° C., at least 5-10 minutes at 80° C., at least 5-10 minutes at 85° C., or at least 5-10 minutes at 90° C.

In some embodiments, the variant phytases have increased thermostability as compared to a parent phytase for at least 5 minutes at 75° C. at pH 5.5, at least 5 minutes at 80° C. at pH 5.5, at least 5 minutes at 85° C. at pH 5.5, or at least 5 minutes at 90° C. at pH 5.5.

Accordingly, as shown in FIGS. 1A-1C, a number of variant phytases of the invention exhibit increased thermostability.

C. pH Stability

In many embodiments, the variant phytases of the invention have increased pH stability at lower pHs, to address the lower pH of the stomach and gastrointestinal tract of non-ruminant animals. That is, many phytases have pH profiles that are suboptimal for the lowered pH environment where the activity is desired in the animal “Increased pH stability” in this context means that the variant enzymes are more stable than the parent phytase (e.g. G1P and/or G7P) under the same pH challenge conditions, that is, the activity of the variant is higher than that of the parental enzyme under identical conditions.

D. Specific Activity Assays

In some embodiments, the variant phytases of the invention have increased specific activity as compared to a parent phytase, particularly G1P and/or G7P. By “specific activity” herein is meant the activity per amount of enzyme, generally determined by dividing the enzymatic activity of a sample (sometimes measure in “phytase units” as discussed herein) by the amount of phytase enzyme, generally determined as is known in the art.

E. Protease Susceptibility

In some embodiments, the variant phytases of the invention are less susceptible to protease degradation than the parent enzyme under identical conditions. In some cases, protease degradation during the production of variant phytases in a production host organism by protease enzymes produced by the host organism can be a problem, thus resulting in lower yield of active enzyme. This is generally determined as is known in the art, for example by allowing proteolytic degradation and then doing N-terminal sequencing on the resulting fragments to determine the cleavage site(s). In some cases, depending on the variant and the host production organism, there may not be significant proteolytic degradation.

As needed, as will be appreciated by those in the art, the specific mutations that can be made will depend on the endogenous proteases that the host organism produces, and also generally occurs in surface exposed loop structures or turns that are therefore accessible to proteases. For example, production of phytases in A. niger fungal production organisms can lead to proteolytic degradation; see Wyss et al., Appl. And Environ. Microbiol. February 1999:359-366, hereby incorporated by reference in its entirety.

IV. Specific Variant Phytases

The present invention provides a number of specific variant phytases with one or more improved properties, specifically thermal stability, and in particular thermal stability at particular temperatures as outlined herein.

Accordingly, the present invention provides variant phytases with one or more improved properties as compared to the wild type G1P sequence (SEQ ID NO:7) and/or G7P (SEQ ID NO:1), wherein the variant phytase is not G7P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have at least 85% identity to G1P (SEQ ID NO:7), with enzymes having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity (but less than 100% identity) to G1P also finding use in the present invention. Accordingly, some embodiments provide variant phytases with from 90% to 99% identity to G1P (SEQ ID NO:7), with other embodiments providing 95% to 99% identity, with the proviso that the phytase is not G1P (SEQ ID NO:7). In some embodiments, the variant phytases of the invention have at least 90% identity to G1P (SEQ ID NO:7). In some embodiments, the variant phytases of the invention have at least 95% identity to G1P (SEQ ID NO:7).

In some embodiments, the variant phytases of the invention have at least 85% identity to G7P (SEQ ID NO:1), with enzymes having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity to G7P also finding use in the present invention, with the proviso that the phytase is not G1P (SEQ ID NO:7) or G7P (SEQ ID NO:1). In some embodiments, the variant phytases of the invention have at least 90% identity to G7P (SEQ ID NO:1). In some embodiments, the variant phytases of the invention have at least 95% identity to G7P (SEQ ID NO:1)

In some embodiments, the invention provides variant phytase enzymes comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1; and wherein said variant phytase enzyme has phytase activity.

In some embodiments, the invention provides variant phytase enzymes comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 2-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.; and wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1. In some embodiments, said variant phytase enzyme has at least 10-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.

In some embodiments, the invention provides variant phytase enzymes comprising one or more amino acid substitutions as compared to SEQ ID NO:1, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 25, 73, 74, 75, 116, 117, 118, 119, 120, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287 and 288 of SEQ ID NO:1, wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:1 and is not SEQ ID NO:1 or SEQ ID NO:7; and wherein said variant phytase enzyme has phytase activity.

In some embodiments, the invention provides variant phytase enzymes comprising one or more amino acid substitutions as compared to SEQ ID NO:1, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 25, 73, 74, 75, 116, 117, 118, 119, 120, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287 and 288 of SEQ ID NO:1, wherein said variant phytase enzyme has at least 2-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.; and wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1. In some embodiments, said variant phytase enzyme has at least 10-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C. In some embodiments, said variant phytase enzyme has at least 2% increase in residual activity as compared to SEQ ID NO:1 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.

In some embodiments, the invention provides the variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said variant phytase enzymes exhibit at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7. In some embodiments, the variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7 exhibit at least 90% sequence identity to SEQ ID NO:7. In some embodiments, the variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7 exhibit at least 95% sequence identity to SEQ ID NO:7.

In some embodiments, the invention provides the variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:1, wherein said variant phytase enzyme exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:1 exhibits at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the variant phytase enzyme as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:1 exhibits at least 95% sequence identity to SEQ ID NO:1.

In some embodiments, the invention provides said variant phytase enzymes as disclosed herein with amino acid substitutions at one of said positions, two of said positions, three of said positions, four of said positions, five of said positions, six of said positions, seven of said positions, eight of said positions, nine of said positions, ten of said positions, eleven of said positions, twelve of said positions, thirteen of said positions, fourteen of said positions, fifteen of said positions, sixteen of said positions, seventeen of said positions, eighteen of said positions, nineteen of said positions, twenty of said positions, twenty-one of said positions, twenty-two of said positions, twenty-three of said positions, twenty-four of said positions, twenty-five of said positions, twenty-six of said positions, twenty-seven of said positions, twenty-eight of said positions, twenty-nine of said positions, thirty of said positions, thirty-one of said positions, thirty-two of said positions, thirty-three of said positions, thirty-four of said positions, thirty-five of said positions, thirty-six of said positions, thirty-seven of said positions, thirty-eight of said positions, thirty-nine of said positions, forty of said positions, forty-one of said positions, forty-two of said positions, or forty-three of said positions of SEQ ID NO:7, wherein the variant phytase enzyme is not SEQ ID NO:1.

In some embodiments, the invention provides said variant phytase enzymes as disclosed herein with amino acid substitutions at one of said positions, two of said positions, three of said positions, four of said positions, five of said positions, six of said positions, seven of said positions, eight of said positions, nine of said positions, ten of said positions, eleven of said positions, twelve of said positions, thirteen of said positions, fourteen of said positions, fifteen of said positions, sixteen of said positions, seventeen of said positions, eighteen of said positions, nineteen of said positions, twenty of said positions, twenty-one of said positions, twenty-two of said positions, twenty-three of said positions, twenty-four of said positions, twenty-five of said positions, twenty-six of said positions, twenty-seven of said positions, twenty-eight of said positions, twenty-nine of said positions, thirty of said positions, thirty-one of said positions, thirty-two of said positions, thirty-three of said positions, thirty-four of said positions, thirty-five of said positions, thirty-six of said positions, thirty-seven of said positions, thirty-eight of said positions, thirty-nine of said positions, forty of said positions, forty-one of said positions, forty-two of said positions, or forty-three of said positions of SEQ ID NO:1, wherein the variant phytase enzyme is not SEQ ID NO:7.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 4 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is P4N.

In some embodiments, the variant phytase has an amino acid substitution of the lysine at position 7 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is K7N.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 25 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is selected from A25F, A25N, and A25Q.

In some embodiments, the variant phytase has an amino acid substitution of the aspartic acid at position 35 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is D35N.

In some embodiments, the variant phytase has an amino acid substitution of the isoleucine at position 55 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is I55V.

In some embodiments, the variant phytase has an amino acid substitution of the histidine at position 60 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is H60Q.

In some embodiments, the variant phytase has an amino acid substitution of the valine at position 67 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is V67A.

In some embodiments, the variant phytase has an amino acid substitution of the Aspartic acid at position 69 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is D69N.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 73 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is A73P.

In some embodiments, the variant phytase has an amino acid substitution of the lysine at position 74 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is K74D.

In some embodiments, the variant phytase has an amino acid substitution of the lysine at position 75 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is K75E.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 116 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is A116Q.

In some embodiments, the variant phytase has an amino acid substitution of the aspartic acid at position 117 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is D117N.

In some embodiments, the variant phytase has an amino acid substitution of the threonine at position 118 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is T118L.

In some embodiments, the variant phytase has an amino acid substitution of the serine at position 119 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is S119E or S119G.

In some embodiments, the variant phytase has an amino acid substitution of the serine at position 120 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is S120K or S120R.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 121 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is P121A.

In some embodiments, the variant phytase has an amino acid substitution of the aspartic acid at position 136 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids namely serine, threonine, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine. In some embodiments, the amino acid substitution is D136S.

In some embodiments, the variant phytase has an amino acid substitution of the asparagine at position 137 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is N137P.

In some embodiments, the variant phytase has an amino acid substitution of the glycine at position 157 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is G157Q.

In some embodiments, the variant phytase has an amino acid substitution of the arginine at position 159 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is R159Y.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 173 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is P173N.

In some embodiments, the variant phytase has an amino acid substitution of the glutamine at position 192 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is Q192N.

In some embodiments, the variant phytase has an amino acid substitution of the serine at position 212 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is S212P.

In some embodiments, the variant phytase has an amino acid substitution of the threonine at position 238 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is T238G.

In some embodiments, the variant phytase has an amino acid substitution of the tyrosine at position 255 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, and valine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is Y255D.

In some embodiments, the variant phytase has an amino acid substitution of the methionine at position 276 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is selected from M276K.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 277 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is A277T.

In some embodiments, the variant phytase has an amino acid substitution of the threonine at position 280 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is selected from T280I, T280L and T280V.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 281 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is P281S or P281T.

In some embodiments, the variant phytase has an amino acid substitution of the histidine at position 282 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is H282N or H282Q.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 283 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is P283G.

In some embodiments, the variant phytase has an amino acid substitution of the proline at position 284 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is selected from P284A, P284S, P284T and P284V.

In some embodiments, the variant phytase has an amino acid substitution of the glutamine at position 285 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is Q285T.

In some embodiments, the variant phytase has an amino acid substitution of the lysine at position 286 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is K286E.

In some embodiments, the variant phytase has an amino acid substitution of the glutamine at position 287 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is Q287N or Q287S.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 288 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is selected from A288H, A288K, A288P, A288R and A288S.

In some embodiments, the variant phytase has an amino acid substitution of the glutamic acid at position 315 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is E315G.

In some embodiments, the variant phytase has an amino acid substitution of the glutamine at position 346 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is Q346S.

In some embodiments, the variant phytase has an amino acid substitution of the phenylalanine at position 354 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is F354Y.

In some embodiments, the variant phytase has an amino acid substitution of the threonine at position 364 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is T364W.

In some embodiments, the variant phytase has an amino acid substitution of the threonine at position 378 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is T378A.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 380 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation). In some embodiments, the amino acid substitution is A380P.

In some embodiments, the variant phytase has an amino acid substitution of the glycine at position 381 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is G381A.

In some embodiments, the variant phytase has an amino acid substitution of the alanine at position 403 of SEQ ID NO:7. In some embodiments, the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects). In some embodiments, the amino acid substitution is A403G.

In some embodiments, the invention provides said variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N, K7N, A25F, A25N, A25Q, D35N, I55V, H60Q, V67A, D69N, A73P, K74D, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, S120R, P121A, D136S, N137P, G157Q, R159Y, P173N, Q192N, S212P, T238G, Y255D, M276K, A277T, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, A288S, E315G, Q346S, F354Y, T364W, T378A, A380P, G381A, and A403G of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N, K7N, A25F, A25N, A25Q, D35N, I55V, H60Q, V67A, D69N, A73P, K74D, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, S120R, P121A, D136S, N137P, G157Q, R159Y, P173N, Q192N, S212P, T238G, Y255D, M276K, A277T, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, A288S, E315G, Q346S, F354Y, T364W, T378A, A380P, G381A, and A403G of SEQ ID NO:7, wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1; and wherein said variant phytase enzyme has phytase activity.

In some embodiments, the invention provides said variant phytase enzymes as disclosed herein comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N, K7N, A25F, A25N, A25Q, D35N, I55V, H60Q, V67A, D69N, A73P, K74D, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, S120R, P121A, D136S, N137P, G157Q, R159Y, P173N, Q192N, S212P, T238G, Y255D, M276K, A277T, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, A288S, E315G, Q346S, F354Y, T364W, T378A, A380P, G381A, and A403G of SEQ ID NO:7, wherein said variant phytase enzyme has at least 2-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.; and wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1; and wherein said variant phytase enzyme has phytase activity.

In some embodiments, the invention provides said variant phytase enzymes comprising the amino acid substitutions as compared to SEQ ID NO:7, wherein the amino acid substitutions are selected from the group consisting of P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288S/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284V/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281T/H282Q/P283G/P284A/Q285T/K286E/Q287N/A288K/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25Q/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25F/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S119G/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/Q192N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, and 212 of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, and S212P of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or two further amino acid substitutions occurring at positions corresponding to positions 74 and/or 192 of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or two further amino acid substitutions K74D and/or Q192N.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 120, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzymes comprising amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.

In some embodiments, the invention provides said variant phytase enzyme comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

In some embodiments, the invention provides said variant phytase enzyme comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5.

In some embodiments, the invention provides said variant phytase enzyme comprising the sequence of SEQ ID NO:3.

In some embodiments, the invention provides said variant phytase enzyme comprising the sequence of SEQ ID NO:5.

In some embodiments, the invention provides variant phytase enzymes having an amino acid substitution set selected from the group consisting of those depicted in FIGS. 1-3 .

In some embodiments, the invention relates to variant phytase enzymes having improved thermal properties, such as thermostability, heat-stability, steam stability, temperature profile, pelleting stability, and/or pH stability.

In some embodiments, the invention relates to variant phytase enzymes having high tolerance to high temperature as compared to G1P (SEQ ID NO:7) and/or G7P (SEQ ID NO:1).

In some embodiments, the invention relates to variant phytase enzymes having improved pelleting stability as compared to G1P (SEQ ID NO:7) and/or G7P (SEQ ID NO:1).

In some embodiments, the invention relates to variant phytase enzymes having improved protease stability as compared to G1P (SEQ ID NO:7) and/or G7P (SEQ ID NO:1), in particular pepsin stability, found in non-ruminant stomachs.

The compositions and methods of the invention thus relates to variant phytase enzymes having improved performance in animal feed (such as an improved release and/or degradation of phytate).

Suitable variant phytases of the invention are for example those depicted in the FIGS. 1-3 .

V. Nucleic Acids of the Invention

The present invention additional provides nucleic acids encoding the variant phytases of the invention. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the variant phytases of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. Thus, providing the amino acid sequence allows the generation of a very large number of different nucleic acid sequences encoding the proteins.

In some embodiments, specific variant phytases are encoded by specific nucleic acid sequences, as are listed in FIGS. 1 and 2 .

As is known in the art, the nucleic acids encoding the components of the invention can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the heterodimeric antibodies of the invention. Generally, the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The expression vectors can be extra-chromosomal or integrating vectors.

The nucleic acids and/or expression vectors of the invention are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with bacteria and fungi finding use in many embodiments.

A. Preparation of Variants

The nucleic acids encoding the variant phytases of the invention can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis and synthetic gene construction as are well known in the art.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips. A preferred technique is GenScript®.

1. Regulatory Sequences

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori phytase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell can be used.

In some embodiments, terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger phytase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

In some embodiments, terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence can also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence can also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

In some embodiments, leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

In some embodiments, suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence can also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the variant-encoding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

In some embodiments, polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger phytase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant phytase being expressed into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant phytase. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant phytase. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger phytase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger phytase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae phytase promoter can be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked with the regulatory sequence.

2. Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used. Vectors contemplated for use with the methods of the invention include both integrating and non-integrating vectors.

In some embodiments, the vector contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.

In some embodiments, the vector contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector can rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector can further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

3. Codon Optimization

Codon optimization can be employed with any of the variant phytase polypeptides of the present invention, in order to optimize expression in the host cell employed. Such methods are well known in the art and described in, for example, WO 2007/142954. In heterologous expression systems, optimization steps can improve the ability of the host to produce the desired variant phytase polypeptides. Protein expression is governed by a host of factors including those that affect transcription, mRNA processing, and stability and initiation of translation. The polynucleotide optimization steps can include steps to improve the ability of the host to produce the foreign protein as well as steps to assist the researcher in efficiently designing expression constructs. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases. The following paragraphs discuss potential problems that may result in reduced heterologous protein expression, and techniques that may overcome these problems.

In some embodiments, reduced heterologous protein expression results from a rare codon-induced translational pause. A rare codon-induced translational pause includes the presence of codons in the polynucleotide of interest that are rarely used in the host organism can have a negative effect on protein translation due to their scarcity in the available tRNA pool. One method of improving optimal translation in the host organism includes performing includes performing codon optimization which can result in rare host codons being modified in the synthetic polynucleotide sequence.

In some embodiments, reduced heterologous protein expression results from by alternate translational initiation. Alternate translational initiation can include a synthetic polynucleotide sequence inadvertently containing motifs capable of functioning as a ribosome binding site (RBS). These sites can result in initiating translation of a truncated protein from a gene-internal site. One method of reducing the possibility of producing a truncated protein, which can be difficult to remove during purification, includes modifying putative internal RBS sequences from an optimized polynucleotide sequence.

In some embodiments, reduced heterologous protein expression occurs through repeat-induced polymerase slippage. Repeat-induced polymerase slippage involves nucleotide sequence repeats that have been shown to cause slippage or stuttering of DNA polymerase which can result in frameshift mutations. Such repeats can also cause slippage of RNA polymerase. In an organism with a high G+C content bias, there can be a higher degree of repeats composed of G or C nucleotide repeats. Therefore, one method of reducing the possibility of inducing RNA polymerase slippage includes altering extended repeats of G or C nucleotides.

In some embodiments, reduced heterologous protein expression occurs through interfering secondary structures. Secondary structures can sequester the RBS sequence or initiation codon and have been correlated to a reduction in protein expression. Stemloop structures can also be involved in transcriptional pausing and attenuation. An optimized polynucleotide sequence can contain minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to allow for improved transcription and translation.

In some embodiments, restriction sites can effect heterologous protein expression. By modifying restriction sites that could interfere with subsequent sub-cloning of transcription units into host expression vectors a polynucleotide sequence can be optimized.

Optimizing a DNA sequence can negatively or positively affect gene expression or protein production. For example, modifying a less-common codon with a more common codon may affect the half life of the mRNA or alter its structure by introducing a secondary structure that interferes with translation of the message. It may therefore be necessary, in certain instances, to alter the optimized message.

AUG or a portion of a gene can be optimized. In some embodiments, the desired modulation of expression is achieved by optimizing essentially the entire gene. In other embodiments, the desired modulation will be achieved by optimizing part but not all of the gene.

The codon usage of any coding sequence can be adjusted to achieve a desired property, for example high levels of expression in a specific cell type. The starting point for such an optimization may be a coding sequence with 100% common codons, or a coding sequence which contains a mixture of common and non-common codons.

Two or more candidate sequences that differ in their codon usage can be generated and tested to determine if they possess the desired property. Candidate sequences can be evaluated by using a computer to search for the presence of regulatory elements, such as silencers or enhancers, and to search for the presence of regions of coding sequence which could be converted into such regulatory elements by an alteration in codon usage. Additional criteria can include enrichment for particular nucleotides, e.g., A, C, G or U, codon bias for a particular amino acid, or the presence or absence of particular mRNA secondary or tertiary structure. Adjustment to the candidate sequence can be made based on a number of such criteria.

Promising candidate sequences are constructed and then evaluated experimentally. Multiple candidates may be evaluated independently of each other, or the process can be iterative, either by using the most promising candidate as a new starting point, or by combining regions of two or more candidates to produce a novel hybrid. Further rounds of modification and evaluation can be included.

Modifying the codon usage of a candidate sequence can result in the creation or destruction of either a positive or negative element. In general, a positive element refers to any element whose alteration or removal from the candidate sequence could result in a decrease in expression of the therapeutic protein, or whose creation could result in an increase in expression of a therapeutic protein. For example, a positive element can include an enhancer, a promoter, a downstream promoter element, a DNA binding site for a positive regulator (e.g., a transcriptional activator), or a sequence responsible for imparting or modifying an mRNA secondary or tertiary structure. A negative element refers to any element whose alteration or removal from the candidate sequence could result in an increase in expression of the therapeutic protein, or whose creation would result in a decrease in expression of the therapeutic protein. A negative element includes a silencer, a DNA binding site for a negative regulator (e.g., a transcriptional repressor), a transcriptional pause site, or a sequence that is responsible for imparting or modifying an mRNA secondary or tertiary structure. In general, a negative element arises more frequently than a positive element. Thus, any change in codon usage that results in an increase in protein expression is more likely to have arisen from the destruction of a negative element rather than the creation of a positive element. In addition, alteration of the candidate sequence is more likely to destroy a positive element than create a positive element. In some embodiments, a candidate sequence is chosen and modified so as to increase the production of a therapeutic protein. The candidate sequence can be modified, e.g., by sequentially altering the codons or by randomly altering the codons in the candidate sequence. A modified candidate sequence is then evaluated by determining the level of expression of the resulting therapeutic protein or by evaluating another parameter, e.g., a parameter correlated to the level of expression. A candidate sequence which produces an increased level of a therapeutic protein as compared to an unaltered candidate sequence is chosen.

In some embodiments, one or a group of codons can be modified, e.g., without reference to protein or message structure and tested. Alternatively, one or more codons can be chosen on a message-level property, e.g., location in a region of predetermined, e.g., high or low GC content, location in a region having a structure such as an enhancer or silencer, location in a region that can be modified to introduce a structure such as an enhancer or silencer, location in a region having, or predicted to have, secondary or tertiary structure, e.g., intra-chain pairing, inter-chain pairing, location in a region lacking, or predicted to lack, secondary or tertiary structure, e.g., intra-chain or inter-chain pairing. A particular modified region is chosen if it produces the desired result.

Methods which systematically generate candidate sequences are useful. For example, one or a group, e.g., a contiguous block of codons, at various positions of a synthetic nucleic acid sequence can be modified with common codons (or with non common codons, if for example, the starting sequence has been optimized) and the resulting sequence evaluated. Candidates can be generated by optimizing (or de-optimizing) a given “window” of codons in the sequence to generate a first candidate, and then moving the window to a new position in the sequence, and optimizing (or de-optimizing) the codons in the new position under the window to provide a second candidate. Candidates can be evaluated by determining the level of expression they provide, or by evaluating another parameter, e.g., a parameter correlated to the level of expression. Some parameters can be evaluated by inspection or computationally, e.g., the possession or lack thereof of high or low GC content; a sequence element such as an enhancer or silencer; secondary or tertiary structure, e.g., intra-chain or inter-chain paring.

In some embodiments, the optimized nucleic acid sequence can express the variant phytase polypeptide of the invention, at a level which is at least 110%, 150%, 200%, 500%, 1,000%, 5,000% or even 10,000% of that expressed by nucleic acid sequence that has not been optimized.

Staring with the amino acid sequence of a variant phytase, a candidate DNA sequence can be designed. During the design of the synthetic DNA sequence, the frequency of codon usage can be compared to the codon usage of the host expression organism and rare host codons can be modified in the synthetic sequence. Additionally, the synthetic candidate DNA sequence can be modified in order to remove undesirable enzyme restriction sites and add or alter any desired signal sequences, linkers or untranslated regions. The synthetic DNA sequence can be analyzed for the presence of secondary structure that may interfere with the translation process, such as G/C repeats and stem-loop structures. Before the candidate DNA sequence is synthesized, the optimized sequence design can be checked to verify that the sequence correctly encodes the desired amino acid sequence. Finally, the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those known in the art.

In some embodiments, the general codon usage in a host organism, such as any of those described herein, can be utilized to optimize the expression of the heterologous polynucleotide sequence in the host organism. The percentage and distribution of codons that rarely would be considered as preferred for a particular amino acid in the host expression system can be evaluated. Values of 5% and 10% usage can be used as cutoff values for the determination of rare codons.

VI. Host Cells and Production Strains

As will be appreciated by those in the art, there are a wide variety of production host organisms for the recombinant production of the variant phytases of the invention, including, but not limited to bacterial cells and fungal cells including yeast. In addition, while the parent phytase is unglycosylated, glycosylation by production in yeast and fungi does not adversely affect the phytase activity.

The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant phytase of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source. In some embodiments, the host cell exhibits transitory expression of the variant phytase. In some embodiments, the host cell is a stably transfected host or a host cell that stably (i.e., permanently) expresses the variant phytase. In some embodiments, the host cell is a production host cell.

The host cell can be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote. Such host cells include but are not limited to bacterial, fungal, and yeast cells. The host cell can also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

The host cell can be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell can be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, BiolTechnology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

VII. Compositions

The present invention also provides compositions comprising a variant phytases. In some embodiments, the composition comprises a carrier and/or an excipient. In some embodiments, the compositions are enriched in such a variant phytase polypeptide of the present invention. The term “enriched” indicates that the phytase activity of the composition has been increased, e.g., with an enrichment factor of at least 1. In some embodiments, the compositions are formulated to provide desirable characteristics such as low color, low odor and acceptable storage stability.

In some embodiments, the composition comprises a variant phytase polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition. In some embodiments, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, alpha-amylase, beta-amylase, phytase, isoamylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, phytase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, polyphenoloxidase, pullulanase, proteolytic enzyme, ribonuclease, transglutaminase, and/or xylanase.

VIII. Methods of Production

The present invention also relates to methods of producing a variant phytase polypeptide, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant phytase polypeptide; and (b) optionally recovering the variant phytase polypeptide.

The host cells are cultivated in a nutrient medium suitable for production of the variant phytase polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant phytase polypeptide is secreted into the nutrient medium, the variant phytase polypeptide can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant phytase polypeptide can be detected using methods known in the art that are specific for the variants. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant phytase polypeptide.

The variant phytase polypeptide can be recovered using methods known in the art. For example, the variant phytase polypeptide can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The variant can be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing the variant is used as a source of the variant. In a particular embodiment, a variant phytase of the invention is not recovered and the host cell is a yeast host cell. In particular, the yeast is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In some embodiments, the yeast is Saccharomyces cerevisiae.

IX. Phytase Formulations and Uses

As discussed herein, the use of phytase in animal feeds has a number of benefits, including a feed cost savings, such as reductions in dietary inorganic phosphate, energy and amino acids, including a fast and efficient breakdown of dietary phytate and increased nutrient availability from phytate, as well as production benefits such as body weight gain for the non-ruminant subjects, the increased release of nutrients from phytate, and a significant benefit in the reduced phosphorus excretion to improve the environmental impacts of non-ruminant animals. In some embodiments, the variant phytases of the invention are formulated and added to feed or can be made as a component of the feed. In the former case, the feed stock addition of phytase can be done by formulating the phytase on a carrier feed such as wheat flour.

As will be appreciated by those in the art, the formulation of the variant phytases of the invention depends on its end use and the associated conditions. Suitable formulations for the variant phytases of the invention include liquid formulations, dried formulations (including spray dried formulations), powdered formulations, granular formulations, and pelleted formulations.

In some embodiments, the enzyme composition (i.e., polypeptide compositions) of the present invention can be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, as a source of the enzymes.

In some embodiments, the enzyme composition can be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.

In some embodiments, the dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

The above compositions are suitable for use in liquefaction, saccharification, and/or fermentation processes, and in some embodiments, in starch conversion. In some embodiments, the compositions are useful for producing a food product, including a syrup, as well as fermentation products, such as ethanol. In some embodiments, the compositions are useful for the pharmaceutical industry, such as digestive aids.

In one embodiment, the phytases are added to animal feed stock and pelleted as is known in the art, such that the feed is formed with phytase in it. In other embodiments, the phytase can be sprayed or dosed in a liquid form into animal feed.

EXAMPLES X. Example 1: EcPhytase G7, G8 and G9 Mutant Design and Construction

The starting gene, G7P, was obtained from the previous EcPhytase patent filing (U.S. patent application Ser. No. 16/566,494). To further improve the stability of G7P against high temperature, eleven, seven and one mutants were designed during G7, G8 and G9 improvements respectively based on analyzing sequence, structural and experimental data. The design includes multiple specific mutations w.r.t. G1P, as shown in FIGS. 1A, 1B and 1C. The mutants were constructed using standard site-directed mutagenesis methods and subsequently cloned into the pYES2-α-factor/CT vector (ThermoFisher Scientific, USA: Catalogue #V8251-20).

XI. Example 2: Preparation of EcPhytase Produced by Saccharomyces cerevisiae in HTP

The Saccharomyces cerevisiae INSCV1 strain (ThermoFisher Scientific, USA: Catalogue #V8251-20) containing recombinant phytase-encoding genes from single colonies were inoculated into individual wells of 96 well plates containing 300 μl synthetic minimal defined medium (SC) with 2% glucose and no uracil supplementation. The cultures were grown overnight at 30° C., 200 rpm and 85% humidity. Appropriate volume of overnight culture from each well needed to obtain an OD600 of 0.4 was added to corresponding wells of the new 96 well plates containing 350 μl of induction medium (SC selective medium containing 2% galactose). The plates were then incubated for 48 hrs at 30° C., 250 rpm and 85% humidity. The cells were then pelleted using centrifugation at 4000 rpm for 10 min at 4° C. The clear supernatants were used to perform the biochemical assays in Example 3 to determine thermostability.

XII. Example 3: Enzymatic Assay to Determine Saccharomyces cerevisiae Produced EcPhytase Thermostability

The supernatants from Example 2 were 100 fold diluted using 0.25M sodium acetate pH 5.5. 50 μl of the diluted supernatants were transferred to PCR plates and heated at 75° C., 80° C., 85° C. and 90° C. for 5 minutes in thermocyclers and subsequently cooled on ice for 2 minutes. In 96 well shallow microtiter plates, 50 μl of the untreated supernatants and heat-treated supernatants were separately added to 100 μl of sodium phytate substrate (C₆H₆Na₁₂O₂₄P₆, FW: 923.81) prepared in 0.25M sodium acetate, pH 5.5. The reaction was incubated at 37° C., 150 rpm for 30 minutes. The reaction was quenched with 100 μl of stop solution (1:2 nitric acid, 100 g/L ammonium molybdate, 2.35 g/L Ammonium vanadate). After the plates were shaked for 30 seconds, they were subjected to plate reader for reading at 415 nm.

The activity of the untreated sample and the heat-treated samples were determined by the activity analysis method mentioned above, and the % residual activity was calculated as Activity_(Heat-treated sample)/Activity_(Untreated sample)×100%.

G7-G9 variants identified with improved Residual Activity after thermochallenge are shown in FIGS. 1A-1C.

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.

All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only. 

1. A composition comprising a variant phytase enzyme comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1; and wherein said variant phytase enzyme has phytase activity.
 2. A composition comprising a variant phytase enzyme comprising one or more amino acid substitutions as compared to SEQ ID NO:7, wherein said one or more amino acid substitutions occur at positions corresponding to positions selected from the group consisting of positions 4, 7, 25, 35, 55, 60, 67, 69, 73, 74, 75, 116, 117, 118, 119, 120, 121, 136, 137, 157, 159, 173, 192, 212, 238, 255, 276, 277, 280, 281, 282, 283, 284, 285, 286, 287, 288, 315, 346, 354, 364, 378, 380, 381, and 403 of SEQ ID NO:7, wherein said variant phytase enzyme has at least 2-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.; and wherein said variant phytase enzyme has at least 85% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1.
 3. The composition of claim 1, wherein said variant phytase enzyme has at least 90% sequence identity to SEQ ID NO:7 and is not SEQ ID NO:1.
 4. The composition of claim 1, wherein said variant phytase enzyme has at least 10-fold increase in residual activity as compared to SEQ ID NO:7 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.
 5. The composition of claim 1, wherein said variant phytase enzyme comprises one or more amino acid substitutions as compared to SEQ ID NO:1 and has at least 90% sequence identity to SEQ ID NO:1.
 6. The composition of claim 1, wherein said variant phytase enzyme has at least 2% increase in residual activity as compared to SEQ ID NO:1 under a condition selected from the group consisting of thermochallenge at about 75° C., thermochallenge at about 80° C., thermochallenge at about 85° C., and thermochallenge at about 90° C.
 7. The composition of claim 1, wherein said one or more amino acid substitution(s) of SEQ ID NO:7 occur at one of said positions, two of said positions, three of said positions, four of said positions, five of said positions, six of said positions, seven of said positions, eight of said positions, nine of said positions, ten of said positions, eleven of said positions, twelve of said positions, thirteen of said positions, fourteen of said positions, fifteen of said positions, sixteen of said positions, seventeen of said positions, eighteen of said positions, nineteen of said positions, twenty of said positions, twenty-one of said positions, twenty-two of said positions, twenty-three of said positions, twenty-four of said positions, twenty-five of said positions, twenty-six of said positions, twenty-seven of said positions, twenty-eight of said positions, twenty-nine of said positions, thirty of said positions, thirty-one of said positions, thirty-two of said positions, thirty-three of said positions, thirty-four of said positions, thirty-five of said positions, thirty-six of said positions, thirty-seven of said positions, thirty-eight of said positions, thirty-nine of said positions, forty of said positions, forty-one of said positions, forty-two of said positions, or forty-three of said positions of SEQ ID NO:7.
 8. The composition of claim 1, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7 or SEQ ID NO:1.
 9. The composition of claim 1, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N, K7N, A25F, A25N, A25Q, D35N, 155V, H60Q, V67A, D69N, A73P, K74D, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, S120R, P121A, D136S, N137P, G157Q, R159Y, P173N, Q192N, S212P, T238G, Y255D, M276K, A277T, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, A288S, E315G, Q346S, F354Y, T364W, T378A, A380P, G381A, and A403G of SEQ ID NO:7.
 10. The composition of claim 9, wherein said one or more amino acid substitutions are selected from the group consisting of substitutions corresponding to P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287S/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284S/Q285T/K286E/Q287N/A288H/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288S/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280I/P281S/H282N/P283G/P284V/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281S/H282N/P283G/P284A/Q285T/K286E/Q287N/A288P/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280V/P281T/H282Q/P283G/P284A/Q285T/K286E/Q287N/A288K/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A250/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/A25F/D35N/155V/H600/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/A73P/K75E/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S119G/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/Q192N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, P4N/K7N/D35N/155V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and P4N/K7N/A25N/D35N/155V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7.
 11. The composition of claim 9, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, and 212 of SEQ ID NO:7.
 12. The composition of claim 11, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, and S212P of SEQ ID NO:7.
 13. The composition of claim 9, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/A25N/D35N/I55V/H60Q/V67A/D69N/A73P/K75E/A116Q/D117N/T118L/S119E/S120K/P121A/D136S/N137P/G157Q/R159Y/P173N/S212P/T238G/Y255D/M276K/A277T/T280L/P281T/H282N/P283G/P284T/Q285T/K286E/Q287N/A288R/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or two further amino acid substitutions occurring at positions corresponding to positions 74 and/or 192 of SEQ ID NO:7.
 14. The composition of claim 13, wherein said one or two further amino acid substitutions are K74D and/or Q192N.
 15. The composition of claim 9, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/S120R/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.
 16. The composition of claim 15, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.
 17. The composition of claim 9, wherein said variant phytase enzyme comprises amino acid substitutions corresponding to P4N/K7N/D35N/I55V/H60Q/V67A/D69N/K74D/P121A/D136S/N137P/G157Q/R159Y/P173N/T238G/Y255D/M276K/A277T/E315G/Q346S/F354Y/T364W/T378A/A380P/G381A/A403G of SEQ ID NO:7, and one or more further amino acid substitutions occurring at positions corresponding to positions selected from the group consisting of 25, 73, 75, 116, 117, 118, 119, 120, 192, 212, 280, 281, 282, 283, 284, 285, 286, 287, and 288 of SEQ ID NO:7.
 18. The composition of claim 17, wherein said one or more further amino acid substitutions are selected from the group consisting of substitutions corresponding to A25F, A25N, A25Q, A73P, K75E, A116Q, D117N, T118L, S119E, S119G, S120K, Q192N, S212P, T280I, T280L, T280V, P281S, P281T, H282N, H282Q, P283G, P284A, P284S, P284T, P284V, Q285T, K286E, Q287N, Q287S, A288H, A288K, A288P, A288R, and A288S of SEQ ID NO:7.
 19. The composition of claim 9, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.
 20. The composition of claim 9, wherein said variant phytase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5.
 21. The composition of claim 19, wherein said variant phytase enzyme comprises the sequence of SEQ ID NO:3.
 22. The composition of claim 20, wherein said variant phytase enzyme comprises the sequence of SEQ ID NO:5.
 23. The composition of claim 1 further comprising animal feed.
 24. A nucleic acid encoding the variant phytase enzyme of claim
 1. 25. The nucleic acid of claim 24 comprises the nucleic acid sequence of SEQ ID NO:4.
 26. The nucleic acid of claim 24 comprises the nucleic acid sequence of SEQ ID NO:6.
 27. An expression vector comprising the nucleic acid of claim
 24. 28. A host cell comprising the nucleic acid of claim
 24. 29. A method of making a variant phytase enzyme comprising culturing the host cell of claim 28 under conditions in which said variant phytase enzyme is produced, and recovering said enzyme.
 30. A host cell comprising the expression vector of claim
 27. 31. A method of making a variant phytase enzyme comprising culturing the host cell of claim 30 under conditions in which said variant phytase enzyme is produced, and recovering said enzyme. 